New biosensors for improved detection of environmental contamination by anticholinesterase pesticides (ACHEB)

Project

QLK3-2000-00650

Cell factory area

3.2.2

EU Contribution

1 459 770 Euro

Duration

36 months

Type

Research project

Starting date

01-09-2000

Keywords

anticholinesterase pesticides

ABSTRACT

Excessive use of pesticides in Europe has led to serious contamination of soil and water and presents a growing threat to human and animal health. We propose to improve the sensitivity of biosensors used both for detecting incidental or chronic environmental contamination by anticholinesterase pesticides and for monitoring the efficiency of remediation of polluted sites. The enzyme acetylcholinesterase is the target of organophosphates and carbamates, two major pesticides currently used in agriculture. We will employ a 'state-of-the-art' approach, utilizing X-ray crystallography, structure analysis, site-directed mutagenesis and large-scale production, using suitable expression systems. This will envable us to produce new bioengineered targets both more sensitive to and more selective for pesticides of interest, and also possessing increased stability. This will permit us to provide our industrial partner with deliverables suitable for production of novel biosensors possessing the specificity and shelf-life required for manufacturing field kits.

OBJECTIVES

We propose here to improve the sensitivity of biosensors presently available for the detection of carbamates (CBs) and organophosphates (OPs). These very common types of pesticides act on phyto- and zoo-parasites through inhibition of acetylcholinesterase, a key enzyme of cholinergic transmission, and usually lead to pest death. CBs and OPs can be harmful for the health of humans, of domestic animals and of the wild fauna either through devastating incidental acute exposure or more discrete chronic contamination by low doses. We will design biosensors using bioengineered AChEs as receptors. They will permit not only a more sensitive detection but also the identification of the anticholinesterase pesticide. These biosensors could also be used to monitor the efficiency of remediation of polluted sites.

DESCRIPTION OF THE WORK

We will continue to explore the molecular diversity of AChEs in new invertebrate species in relation to their susceptibility to OPs and CBs. Structural models of the active site of these enzymes will be derived using the crystallographic data obtained recently by one group of the consortium on Drosophila AchE and which are expected on other invertebrate AChEs during this project. Precise structural 3-D models will be used as guidelines for directed mutagenesis in order to increase AchE sensitivity or specificity to certain agents and the overall stability of the molecule. The normal attachment means of AChEs will also be manipulated by directed mutagenesis to improve the attachment of these new targets on artificial membranes. This part of the work will enable us to provide our partners specialized in biosensor design with a large number of AChE muteins with controlled inhibitor sensitivity.

The technological development of our project has two major goals. We intend first to design one product which will be brought to the market. The On Site Tester (OST) will use the technology of Affinity Enzymo Metric Assay (AEMA) with a single Ache mutein of high sensitivity for improved detection (OST). In parallel to this production, we will also develop a new generation of biosensor prototypes by improving the technology of multielectrodes biosensors. Prototypes will possess a hundred different AChE muteins and as many electrodes (Mutein Arrays chip). Signals will be analysed by an Artificial Neural Network. No industrial development of these latter prototypes is planned within this project.

DELIVERABLES

Crystallization and X-ray analysis of new insect and of nematode AChEs are expected, leading to high quality 3D models of the active gorge in these species (public dissemination through scientific papers). Optimisation of in vitro production of bioengineered AChEs with increased sensitivity to inhibitors and stability will be achieved (Public dissemination of protocols).

Further structural studies on non catalytic suunits mediating AChE attachment to membranes (for application to attachment of enzymes in biosensors).

An on site tester, dedicated for field use, will be developed in this project using molecular fusions of AChE and several enzyme markers (patents). This product will be marketed by our industrial partner. A prototype combining hundred of AChE muteins on a membrane (mutein array chips) will also be patented.